1. Field of Invention
The present invention relates to the field of environmental restoration technique for soil with organic toxicants pollution, specifically as a method of enhanced remediation of petroleum contaminated soil by utilizing the combination method of an ornamental plant Impatiens balsamina L. and mixed hydrocarbon degraders including bacteria and fungi.
2. Description of Related Arts
Soil plays an essential part in the ecosystem and is one of the environmental resources upon which human depend. In the last decades, however, with the increasing types and amounts of toxic organics, more and more organic contaminants transport and enter into the soil environment. The organic contaminants in soil would not only lead to the decay of organisms, but also endanger human health and even ecological safety through the food chain. Furthermore it could bring pollution of surface and subsurface water due to contaminants transportation, which consequently threatens the people's living environment and even exposes human beings to the greater environmental risks. (See Reference 1: Fismes, J.; Perrin-Ganier, C.; Empereur-Bissonnet, P. Soil-to-Root Transfer and Translocation of Polycyclic Aromatic Hydrocarbons by Vegetables Grown on Industrial Contaminated Soils. J. Environ. Qual. 2002, 31, 1649-1656; Reference 2: Zhou Qixing, Kong Fanxiang and Zhu Lin. Ecotoxicology. Beijing, Chemical Industry Press. 2005, 119-144) Therefore, remediation of toxic organic contaminants in soil has been one of the hot research fields in soil and environmental science all around the world. (See Reference 4: Chaudhry, Q; Blom-zanddstra, M.; Gupta, S.; Erick J. Joner. Utilising the synergy between plants and rhizosphere microorganisms to enhance breakdown of organic pollutants in the environment. Environ. Sci. & Pollut. Res. 12 (1) 34-48; Reference 5: Euliss K. Ho C H. Schwab A P. et al. 2008. Greenhouse and field assessment of phytoremediation for petroleum contaminants in a riparian zone. Bioresource Technology, 99:1961-1971; Reference 6: Song Yufang, Xu Huaxia, Ren Liping, Bioremediation of mineral oil and polycyclic aromatic hydrocarbons (PAHs) in soils with two plant species[J]. Chinese Journal of Applied Ecology, 2001, (1):108-112). The generated oily mud, cuttings and ground crude oil during the processes of oil exploration and production, storage and transportation could cause serious pollution in a large area in the vicinity of wells. The excess petroleum hydrocarbon (PHC) contaminants in soils will destroy soil structure, scatter earth grain, and reduce the water penetration in soil. What's more, the PHC polluted soil has high hydrophobicity, which will result in the reduction of moisture absorption and water storage, thereafter the plant growth inhibition in it. (See Reference 7: Liste H H. Felgentreu D. 2006. Crop growth, culturable bacteria, and degradation of petrol hydrocarbons (PHCs) in a long-term contaminated field soil. Applied Soil Ecology, 31: 43-52.) The subsurface water could be also polluted due to the infiltration of PHC. (See Reference 8: N. Sawatsky, X. Li. Importance of soil-water relations in assessing the endpoint of bioremediated soils. Plant and Soil. 1997, 192: 227-236.) Therefore, remediation of PHC contaminated soil is necessary and urgent.
Remediation of PHC contaminated soil proves to be difficult in both the academic and practical fields. Present remediation methods include physical-chemical method, and bioremediation method. The conventional physical-chemical method has a high cost and easily results in the secondary pollution, thus it is not suitable for large-scale application. It is noticeable that the microorganism might bring biological pollution to local soils and subsurface water resources. In terms of the organic contaminants which are difficult to be degraded by microorganisms, the bioremediation method cannot be used. Therefore, the safe, reliable and cost-effective new technique will play a more important role in the remediation of organic contaminated soil. (See Reference 9: Dai Shugui, Progress in environmental chemistry, Beijing: Chemical Industry Press: 2005, 119-120). As a new environmental restoration technique, phytoremediation of organic contaminated soil is to in-situ remediate the contaminated soil by adsorbing, degrading and passivating organic contaminants during the plant growth. (See Reference 10: Zhou Qixing, Song Yufang, 2004, principles and methods for remediation of contaminated soil, Beijing: Science press) Compared with the conventional method, the main advantages of phytoremediation of organic contaminated soil include minimal environmental damage, no site disposal, treatment of various toxic substances simultaneously, cost effectiveness, aesthetically pleasing and high public acceptance. (See Reference 11: Macek T. et al. 2000. Biotechnology Advances. 18:23; Reference 12: Euliss K. Ho C H. Schwab A P. et al. 2008. Greenhouse and field assessment of phytoremediation for petroleum contaminants in a riparian zone. Bioresource Technology, 99:1961-1971; Reference 13: Schnoor J L. Phytoremediation Technology Evalution Report, Prepared for Groundwater Remediation Technologies Analysis Center. http://gwrtac.org. 1997; Reference 14: Dai Shugui, Progress in environmental chemistry, Beijing: Chemical Industry Press: 2005, 119-122) Therefore, phytoremediation becomes one of the hot research topics in the remediation field.
When applying the phytoremediation technique, selection of a suitable plant degrading petroleum hydrocarbons in soils is the essential prerequisite and foundation. So far, however, there are few studies on the selection of plants capable to remediate PHCs in soils around the world. The plants with intellectual property used for remediation of PHC contaminated soil are still less. Furthermore, due to the insufficient understanding on the agronomic characters, pest control, breeding potential and physiological characters, the application of phytoremediation has been impeded. (See, Reference 15: Ute K. Phytoremediation: novel approaches to cleaning up polluted soils. Current Opinion in Biotechnology, 2005, 16:133-141.) Therefore, screening more effective species for phytoremediation of PHC contaminated soil is still an important task.
Ornamental plant is a plant group with a long history of utilization, which includes over 400,000 cultivated varieties. China is one of the countries which have the most abundant varieties of ornamental plants in the world. (See References 16, Guo Weiming, Mao Longsheng. Introduction to ornamental horticulture [M]. China Agriculture Press, 2001) Selection of hyperaccumulators from the ornamentals for phytoremediation has the following feasibilities and advantages including: (1) The ornamental plant has abundant species including both the herbaceous and woody plant, which establishes a firm foundation for screening. (2) The ornamental plant can beautify the environment while degrading the pollution, which is killing two birds with one stone. (3) The ornamental plant usually does not enter the food chain, which prevents potential hazards from entering human body. (4) Many ornamental plants have certain economic value, and can serve as the raw materials therefore decreasing the remediation cost. (5) The ornamental plant has a long history of cultivation and the cultivation system has been well established and highly mechanized in China, which guarantee the remediation practice using ornamental plants. (See Reference 17: Liu, J.-n., Zhou, Q.-x., Sun, T., et al. Growth responses of three ornamental plants to Cd and Cd—Pb stress and their metal accumulation characteristics. Journal of Hazardous Materials, 2008, 151(1): 261-267.). Therefore, it is entirely practicable to screen ornamental plants with high PHCs-degrading potentialities, and to apply the plants to the subsequent remediation practice.
The plant-microorganism combined effect can improve the PHC remediation efficiency. The higher plants and their associated rhizosphere microorganisms in the organic contaminated soil with mutual benefits to each other. The higher plants release the photosynthesis products into the soil by the roots. For example, the required nutrients and oxygen for the growth and propagation of the microorganism released by plants can increase the activity and quantity of the microorganisms, improve the diversity of microbial community structures, so as to assist the microorganism in degrading the target organic contaminants more effectively and efficiently.
In return the rhizosphere microorganism secretes chemical substances which can help the plants alleviate the toxic effect of contaminants on the plant roots, so as to help the plant grow healthier and better degrade contaminants. The rhizosphere of the higher plant provides the microorganism with a space suitable for growth and propagation. In the rhizosphere, the combined action of the plant roots and the microorganisms can accelerate the degradation of the organic contaminants, especially those can hardly be degraded by conventional methods.
Plant Growth Promoting Rhizobacteria (PGPR) refers to microorganisms capable of promoting the absorption and utilization of mineral nutrition by the plant, or improving the biomass of the plant, or even refers to microorganisms having an inhibiting effect on the harmful microorganisms. The PGPR usually refers to bacteria which have capability to fix nitrogen, solubilize phosphate and decompose potassium or have function of generating plant hormone. (See Reference 18: Kloepper, J. W., et al., Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. Nature 1980. 286: p. 885-886. Reference 19: Kloepper, J. W. and M. N. Schroth, Relationship of in vitro antibiosis of plant growth-promoting rhizobacteria to plant growth and the displacement of root microflora. Phytopathology, 1981. 71: p. 1020-1024.) The contaminants in soil can make plants generate ethylene which will inhibit the plant growth. However, the PGPR can produce indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. The ACC deaminase can decompose ACC, therefore protecting the plant and enhancing growth. (See Reference 20 Glick, B. R., Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase. FEMS Microbiology Letters, 2005. 251(1): p. 1-7). The reduction of ethylene makes the plant grow better in the contaminated environment and exerts a vital role in the remediation of contaminated soil. In addition, the PGPR serves as the biocontrol bacteria to protect the rhizosphere from the virus invasion. (See Reference 21: Compant, S., et al., Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Applied and Environmental Microbiology, 2005. 71(9): p. 4951-4959.)
Euliss et al. suggested screening and combining different microorganisms and special plant for enhanced remediation of PHC contaminated soil. (See Reference 22: Liste, H. H. and D. Felgentreu, Crop growth, culturable bacteria, and degradation of petrol hydrocarbons (PHCs) in a long-term contaminated field soil. Applied Soil Ecology, 2006. 31(1-2): p. 43-52.)
Song Yufang et al. discovered that PHC degradation efficiency by microorganisms separated from rhizosphere is much higher than those separated from blank soils. Thus inoculating microorganism can not only protect plants from damage by contaminants but also improve degradation efficiency of PHC. Recent study indicates that inoculating microorganism, especially indigenous microorganism, into contaminated soil can significantly improve the remediation efficiency. Moreover, the indigenous microorganisms adapt easily to the local environment without competing with the local species. (See Reference 23: Atlas, R. M. and R. Bartha, Microbial Ecology: Fundamentals and Applications. 4th ed. 1998, Redwood City, Calif.: Cummings Science Publishing.)
The previous studies are mainly focused on inoculating the PHC degrading bacteria in the soil for enhancing the remediation efficiency. However, the plant-fungi combined method for remediating PHC contaminated soil also has a good efficiency. (See Reference 24: Hashem, A. R., Bioremediation of petroleum contaminated soils in the Arabian Gulf region: a review. J. Kuwait Sci., 2007. 19: p. 81-91). Based on the high PHC degradation efficiency of ornamental plant Impatiens balsamina L., the present invention studies and analyzes the technique of plant-microorganism combined method for remediation of petroleum contaminated soil. The remediation technique adopts the following materials: higher plant Impatiens balsamina L. which has been screened by us before, 3 strains of indigenous bacteria for degrading PHCs: {circle around (1)} Bacillus subtilis, {circle around (2)} Bacillus licheniformis and {circle around (3)} Bacillus polymyxa, and 2 strains of indigenous fungi for degrading PHCs: {circle around (1)} Trichosporon behrend, and {circle around (2)} Candida tropicalis. Four treatments are applied as follows:
(1) single treatment by planting Impatiens balsamina L.;
(2) planting Impatiens balsamina L. with inoculating solution of petroleum hydrocarbon degrading microorganisms;
(3) planting Impatiens balsamina L. with inoculating solution of plant growth-promoting rhizobacteria (i.e. azotobacter, phosphorus-solubilizing bacteria, potassium bacteria); and
(4) planting Impatiens balsamina L. with inoculating solution of petroleum hydrocarbon degrading bacteria and inoculating solution of plant growth-promoting rhizobacteria.
An optimum result will be selected from the treatments mentioned above.